Abstract Schistosomiasis is a parasitic disease of poverty caused by a bloodfluke that is transmitted by certain species of freshwater snails. Over 240 million people are infected and as many as 800 million are at risk, principally in sub-Saharan Africa. Parasite eggs that are lodged in the visceral organs induce inflammation and fibrosis leading to chronic pain, malaise and fatigue. If left untreated, the disease impairs the school performance of children and worker productivity which, in subsistence communities, undermines social and economic development. Treatment of schistosomiasis relies on just one drug, praziquantel (PZQ) and in the absence of backup drugs there are concerns regarding drug resistance. Also, PZQ is not particularly effective at the single oral dose administered. The WHO has declared that new therapies are needed to treat schistosomiasis. Parasites produce proteases (proteolytic enzymes) that are essential to their survival, growth and reproduction in the host. One protease, known as the proteasome, is a multi-subunit protein complex that regulates normal protein turnover and degradation of misfolded proteins in eukaryotic cells. Recent high profile studies, including one by the PI, have shown that proteasome inhibitors that are selective for Plasmodium, Trypanosoma or Leishmania species, can either eliminate or reduce parasite burden by more than 95% in animal infection models. For our study, we employed a mass spectrometry-based, substrate specificity profiling strategy to direct the design of a potent and selective anti-malarial proteasome inhibitor. Here we hypothesize that the proteasome of the schistosome parasite is a suitable drug target. In support of this, the preliminary data demonstrate that exposure of Schistosoma mansoni in culture to a selection of diverse proteasome inhibitors immobilizes the parasite and causes deleterious phenotypic changes. The data also show that the inhibitors engage the proteasome (Sm20S) target in living parasites. The inhibitors employed so far lack selectivity for Sm20S over the human constitutive proteasome (c20S) and immunoproteasome (i20S). However, our preliminary data show that the peptide cleavage specificity of Sm20S is sufficiently different from those of c20S and i20S to indicate that selective inhibitors of Sm20S could be developed. In Aim 1, therefore, we will purify Sm20S from S. mansoni and employ our mass spectrometry based, substrate profiling method to define the Sm20S cleavage specificity in relation to c20S and i20S. This specificity information will be used to synthesize peptidyl inhibitors specific to Sm20S using the potent carmaphycin B inhibitor as a scaffold. In Aim 2, we will screen three high-value proteasome inhibitor collections from industrial and academic collaborators. In Aim 3, we will evaluate the potency of hit compounds in S. mansoni culture and in other relevant schistosome species, confirm engagement of the proteasome target and evaluate cytotoxicity against mammalian cell lines. The proposed project will define a set of pre-validated proteasome inhibitors of the schistosome proteasome that can be taken forward into an expanded pre-clinical development program.